J. Biol. Chem., Vol. 262, Issue 11, 5015-5019, Apr, 1987
Energy-linked nicotinamide-nucleotide transhydrogenase. Light-driven transhydrogenase catalyzed by transhydrogenase from beef heart mitochondria reconstituted with bacteriorhodopsin
GD Eytan, E Eytan and J Rydstrom
Purified nicotinamide-nucleotide transhydrogenase from beef heart
mitochondria was co-reconstituted with bacteriorhodopsin to from
transhydrogenase-bacteriorhodopsin vesicles that catalyze a 20-fold
light-dependent and uncoupler-sensitive stimulation of the reduction of
NADP+ and NADP+ analogs by NADH and a 50-fold shift of the nicotinamide
nucleotide ratio. In the presence of light, the transhydrogenase-
bacteriorhodopsin vesicles catalyzed a pronounced light intensity-
dependent inward proton pumping as indicated by a pH shift of the medium.
As indicated by pH shifts, proton pumping by the bacteriorhodopsin
essentially paralleled the light-driven transhydrogenase. Addition of
valinomycin increased the pH shift twice with a concomitant 50% inhibition
of the light-driven transhydrogenase, whereas nigericin inhibited the pH
shift completely and the light- driven transhydrogenase partially. Taken
together, these results suggest that transhydrogenase and bacteriorhodopsin
interact through a delocalized proton-motive force. Possible partial
reactions of transhydrogenase were investigated with transhydrogenase-
bacteriorhodopsin vesicles energized by light. Reduction of oxidized 3-
acetylpyridine adenine dinucleotide by NADH, previously claimed to
represent partial reactions, was found to require NADPH. Similarly,
reduction of thio-NADP+ by NADPH required NADH. It is concluded that these
reactions do not represent partial reactions.
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